Material required for the analyses

• Material for preparing the sample and serial dilu-tions, described in Chapter 2.

• The culture medium recommended for the test to be carried out, described in specific chapters.

• Sterile, empty 20 × 100 mm Petri dishes.

• Laboratory incubator set to the temperature speci-fied by the test to be performed, described in spe-cific chapters.

3.2.2 Procedure

Before starting the procedure, observe the precautions and care described in Chapter 2, to ensure that all activi-ties will be carried out under aseptic conditions. Prop-erly identify the tubes and plates that will be inoculated by labeling them with the sample code, the dilution, and the standard abbreviation of the culture medium.

Melt the culture media in a boiling water bath, main-taining the boiling for only the time necessary to soften and liquefy the agar. Cool immediately in cold water and keep at a temperature of 44 to 46°C until the time of use (in a temperature-controlled water bath or incubator).

a) Preparation of the samples and serial dilu-tions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2.

b) Inoculation: In general, inoculation is done for several tests at the same time. For each test that is being conducted, select three adequate dilutions of the samples (see the notes below) and inocu-late 1 ml of each dilution in separate, sterile and empty Petri dish, opening the plates only enough to insert the pipette. Work in a laminar flow

cabi-net or in the proximity of the flame of a Bunsen burner. Deposit the inoculum off-centre in the Petri dish, since this will later on facilitate mixing with the culture medium. Position the pipette at an angle of about 45° touching the bottom of the plate. Use a different pipette for each dilution, with a maximum holding capacity of 10 ml. The uncertainty of the volume measurement must not exceed 5% (ISO 6887-1:1999). Observe carefully whether the plate used actually corresponds to the sample and dilution that are being inoculated.

Change the position of the plates as they are being inoculated, to avoid the risk of inoculating the same plate more than one time, or to leave a plate un-inoculated.

Note b.1) Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. If the expected contamination level of the inoculum falls in range from 2.500 to 25.000 CFU/g or ml, for example, the recommended dilutions are 10⁻¹, 10⁻² and 10⁻³, which correspond to 0.1, 0.01 and 0.001 g or ml of sample. If the contamination level is expected to exceed this range, higher dilutions must be inocu-lated. If the contamination is expected to be below this range, it is possible to start inoculating 1 ml of the sample without any dilution for liquid prod-ucts. In the case of solid products it is not possible to inoculate samples without dilution, but it is pos-sible to inoculate up to 2 ml of the initial dilution in one and the same plate, or a greater volume, dis-tributed or divided over several plates (2 ml/plate).

If it is not possible to previously estimate the level of contamination of the sample, it is recommended to inoculate more than three dilutions, made from the initial dilution.

Note b.2) For the analysis of certain foods, the recommended initial dilution is greater than 1:10 (see Chapter 2).

If the counts in these products are expected to be low, the inoculated volume of the initial dilution should be increased, in the same way as described in Note b.1. When using this technique, the inocu-lation of 0.1 g of solid product or 1 ml of liquid product should be maintained, if possible.

Note b.3) To increase the accuracy of the counts, it is recom-mended not to use pipettes with a holding capacity greater than 2 ml to dispense volumes of 1 ml. It is also possible to inoculate two plates with the same dilution (duplicate).

Note b.4) ISO 7218:2007 does not require the inoculation of three dilutions of the sample, nor that of two plates for each dilution. It establishes two successive dilu-tions, without duplicate, or with a duplicate if only one dilution is to be used. However, the way to

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culate the results is somewhat different (see item 3.7).

c) Addition of the culture medium: For each test that is being conducted, withdraw the culture medium from the water bath or incubator at 44–46°C and, if the flask is wet, dry with a paper towel taking care to avoid spattering onto the plates at the moment of plating. Avoid agitation and abrupt movements to prevent the formation of bubbles. Pour 12 to 15 ml of the medium into the inoculated plates, observing whether the iden-tification of the plates corresponds to the culture medium used. Mix the medium with the inocu-lum, moving the plates gently on a flat surface, in movements forming the number eight or in cir-cular movements, 8–10 times clockwise and 8–10 times counter-clockwise. The plates should be moved about with utmost care, to avoid droplets of medium from spattering onto the rims or lids of the plates. To facilitate this step of the operation, prefer using high plates (20 × 100 mm). The plates can be stacked one on top of the other during the addition of the growth medium and homogeni-zation with the inoculum, but they must subse-quently be evenly distributed over the cold surface of a bench, to accelerate cooling and solidification of the medium.

Note c.1) When several tests are being conducted simulta-neously, the activities and teamwork should be organized and programmed so as to satisfy the fol-lowing conditions, established by the Compendium (Swanson et al., 2001): the time between deposit-ing the inoculum in a plate and the addition of culture medium should not exceed 10 minutes, to prevent the inoculum from drying out and to adhere to the plates. Mixing the culture medium with the inoculum should be done immediately after adding the medium, in order to avoid the risk of solidification of the agar. Furthermore, the Compendium recommends that the complete pro-cedure, from the preparation of the first dilution until finishing the inoculation of all the culture media, should not take longer than 20 min-utes. ISO 6887-1:1999 recommends not exceed 45 minutes.

Note c.2) For foods containing or consisting of particles (meals and flours, for example) the distinction between colonies and particles may be difficult in the first dilution. To avoid this problem, TTC (2,3,5 triphenyltetrazolium chloride) may be added to the culture medium since most bacteria form red colonies in the presence of TTC. For each 100 ml

of medium, add 0.5 ml of a 1% aqueous TTC solu-tion, previously sterilized by filtration.

d) Incubation: Wait until solidification of the culture medium is completed, invert the plates (if required by the method being used) and incubate at the conditions of temperature, time and atmosphere specified for each test. The culture medium should reach the incubation temperature within a maxi-mum time interval of two hours. Avoid excessive stacking of the plates and do not place an exces-sive number of plates in each incubator, to ensure even distribution of the temperature. Within the first 48 h of incubation, the plates may not lose more than 15% of their weight caused by drying out. Excessive moisture is also undesirable, since it increases the risk of spreading. Depending on the temperature, humidity control of the incubator may be necessary.

e) Counting the colonies and calculating the results:

Follow the guidelines and instructions described in item 3.6.1.

3.3 Spread plate technique

The main difference between surface plate and pour plate is that the sample and/or its dilutions are inocu-lated directly onto the surface of a solid medium, previ-ously distributed over a certain number of Petri dishes.

Surface inoculation is considered advantageous in some aspects, since it does not expose the microorganisms to the high temperature of the melted medium, allows visualization of the morphological and differential char-acteristics of the colonies, facilitates the transferring of colonies, allows to use media that may not be re-heated to melt the agar, and does not require that the culture media be transparent or translucent. Its main disadvan-tage is the volume to be inoculated, which is limited to the maximum liquid absorption capacity of the culture medium (0.5 ml per plate). The standard procedure is the inoculation of 0.1 ml/plate of each dilution, with a detection limit of 100 CFU/g for solid products or 10 CFU/ml for liquid products. This procedure can be adapted, if necessary, to a detection limit of 10 CFU/g for solid products or 1 CFU/ml for liquid products.

Its main applications are total aerobic psychrotrophic counts, yeast and mold counts, S. aureus counts and B. cereus counts.

3.3.1 Material required for the analyses

• Materials for preparing the sample and serial dilu-tions, described in Chapter 2.

• Petri dishes containing the medium recommended for the test, described in specific chapters.

• Glass or plastic spreaders (Drigalski) immersed in ethanol 70%.

• Laboratory incubator with the temperature set at the temperature specified by the test to be performed, described in specific chapters.

3.3.2 Procedure

As recommended for pour plate, observe the precautions and care described in Chapter 2 before starting the pro-cedure, to ensure that all activities be carried out under aseptic conditions. Identify all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium.

Prepare the plates on beforehand and dry them in a lam-inar flow cabinet for 30–60 min with the lids partially open or in an incubator at 50°C/1.5–2 h with the lids partially open or in an incubator at 25–30°C/18–24 h with the lids closed.

a) Preparation of the samples and serial dilu-tions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2.

b) Inoculation: In general, inoculation is done for several tests at the same time. For each test that is being conducted, select three adequate dilu-tions of the sample to be inoculated (see note b.1).

Using a pipette with a maximum holding capac-ity of 1 ml (and 0.1 ml graduation markings), inoculate 0.1 ml of each dilution onto the surface of previously prepared plates. Verify whether the identification of the plate actually corresponds to the sample and dilution that are being inoculated and whether the plate contains the correct culture medium. Change the position of the plates as they are being inoculated, to avoid the risk of inocu-lating the same plate more than one time, or to leave a plate un-inoculated. Work in a laminar flow cabinet or in the proximity of the flame of a Bun-sen burner. Spread the inoculum onto the entire

surface of the medium as fast as possible, using glass or plastic spreader (Drigalski), and continue until all excess liquid is absorbed. Utilize a differ-ent spreader for each plate or, alternatively, flame-sterilize the spreader after each plate, starting with the greatest dilution plate and going to the smallest dilution plates.

Note b.1) When several tests are being performed simulta-neously, the activities and teamwork should be organized and programmed so as to satisfy the following conditions, established by Compendium (Swanson et al., 2001): the complete procedure, from the preparation of the first dilution until finishing the inoculation of all the culture media, should not take longer than 20 minutes and the inoculum spreading should be started immedi-ately after depositing the three dilutions onto the medium surface. ISO 6887-1:1999 recommends not exceed 45 minutes.

Note b.2) As described for pour plating, select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. However, it should be taken into account that the inoculated volume is ten times smaller. In the case of samples with a low level of contamination, a greater volume of the first dilu-tion may be inoculated, distributing this volume over several plates. A distribution commonly used is inoculating three plates with 0.3 ml and one plate with 0.1 ml. The spreading of 0.3 ml onto the plates requires a longer time of absorp-tion of the liquid, thus care and precauabsorp-tions must be taken to avoid that moisture films remain on the surface, with the consequent formation of spreading zones.

Note b.3) For the analysis of certain foods, the recom-mended initial dilution is greater than 1:10 (see Chapter 2). If the expected counts in these prod-ucts are low, the inoculated volume of the initial dilution should be increased, in the same way as described in Note b.1. When using this technique the inoculation of 0.01 g of solid products or 0.1 ml of liquid products should be maintained, if possible.

Note b.4) To increase the accuracy of the counts, it is rec-ommended not to utilize pipettes with a capacity greater than 1 ml to dispense volumes of 0.1 ml.

Note b.5) ISO 7218:2007 does not require the inoculation of three dilutions of the sample, establishing only two successive dilutions, without duplicate, or with a duplicate if only one dilution is to be used. How-ever, the way to calculate the results is somewhat different (see item 3.7).

c) Incubation: Follow the same instructions and guidelines as those described for pour plating.

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d) Counting the colonies and calculating the results:

Follow the guidelines and instructions described in item 3.6.2.

3.4 Drop plate technique

The drop plate method is a surface inoculation tech-nique that has the same advantages as the spread plating technique. The main difference is that the inoculum is not spread, but deposited onto the surface of the cul-ture medium in the form of 0.01 ml-droplets. Since the droplets occupy a minimum amount of space, it is possible, on one and the same plate, to inoculate three dilutions in triplicate, three drops per dilution. This makes this technique extremely cost-friendly, with a detection limit 1,000 CFU/g for solid products or 100 CFU/ml for liquid products. Although the drop plate technique is not routinely used for the micro-biological examination of foods, it can be very useful in situations that require the inoculation of a large number of dilutions.

3.4.1 Material required for the analyses

• Materials for preparing the sample and serial dilu-tions, described in Chapter 2.

• Sterile 0.1 ml graduated pipettes or pipettes with disposable tips to dispense the droplets.

• Petri plates containing the medium recommended for the test, described in specific chapters.

• A laboratory incubator with the temperature set at the temperature specified by the test to be per-formed, described in specific chapters.

3.4.2 Procedure

As recommended for pour plate, observe the precau-tions and care described in Chapter 2 before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling all the tubes and plates that will be inoculated with the sam-ple code, the dilution and the standard abbreviation of the culture medium. Prepare the plates with the culture medium on beforehand and leave to dry in an incubator for 24 hours at 25–30°C with the lids closed.

a) Preparation of the samples and serial dilu-tions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2, but prepare the diluent supplemented with 0.1% agar, to make it easier to fix the droplets later on onto the surface of the culture medium.

b) Inoculation: Divide the plate into nine sectors, marking the bottom with a glass-marking pen (trac-ing three horizontal lines and three vertical lines). For each test that is being conducted, select three adequate dilutions of the sample to be inoculated (see note b.1 below). Before collecting the volume of each dilution to be inoculated onto the plates, vigorously agitate or shake the dilution tubes, by inverting them 25 times in an 30 cm-arc or with the aid of a vortex shaker.

Using 0.1 ml (and 0.01 ml graduated) pipettes or pipettes with disposable tips, deposit three 0.01 ml drops of each dilution in three adjacent quadrates of the plate (triplicate). This procedure must be per-formed with care, to avoid any droplets from running out of their respective quadrate. Do not spread the drops. Keep the plates placed on a flat surface, wait until all the liquid is absorbed by the culture medium, which will require approximately 30 minutes.

Note b.1) Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain drops containing 30 colonies, at most. Take into account that drop plating cannot be used with samples with a contamination level lower than 10³ CFU/g or 10² CFU/ml, except when the pur-pose of the test is not to quantify, but rather to show or prove that the count is below this limit.

c) Incubation: Wait until the liquid of the drops is completely absorbed by the culture medium and incubate under the same conditions recommended for pour plating.

d) Counting the colonies and calculating the results:

Follow the guidelines and instructions described in item 3.6.3.

3.5 Membrane filtration

The procedure of membrane filtration is limited to the examination of limpid or crystal-clear liquid sam-ples, without solids in suspension, and which may be filtered through a membrane with a pore size of 0.45 μm. The main advantage of this technique is that it makes it possible to inoculate larger volumes

of the sample, concentrating in the membrane the microorganisms present in the inoculated quantity.

The detection limit is 1 CFU per inoculated volume, which makes it the technique of choice for examining samples containing counts lower than the detection limit of the other procedures. Its main applications are total aerobic mesophilic counts, yeast and mold counts, lactic acid bacteria counts, enterococci counts and counts of total coliforms, fecal coliforms and E. coli in water, carbonated soft drinks and other liq-uid products, in addition to solid products, provided they can be transformed into a limpid solution, such as salt and sugar.

3.5.1 Material required for the analyses

• Material for preparing the sample and serial dilu-tions, described in Chapter 2.

• A previously sterilized filtration set.

• A vacuum pump.

• Membrane-filters, 47 mm in diameter, porosity of 0.45 μm.

• Petri dishes containing the culture medium recom-mended for the test, described in specific chapters.

• Sterile empty Petri dishes and sterile pads, optional for use with broth media.

• A laboratory incubator with the temperature set at the temperature specified by the test to be per-formed, described in specific chapters.

3.5.2 Procedure

As recommended for pour plating, observe the precau-tions and care described in Chapter 2 before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium.

a) Preparation of the filtration set: The membrane filtration set is composed of a membrane filter holder, a kitasato flask and a filtration cup. The filter holder is a kind of funnel the upper part of which is plane, to accommodate the filtration membrane and onto the top of which the filtration

a) Preparation of the filtration set: The membrane filtration set is composed of a membrane filter holder, a kitasato flask and a filtration cup. The filter holder is a kind of funnel the upper part of which is plane, to accommodate the filtration membrane and onto the top of which the filtration